Extensive research in apoptosis has led to the identification of the core components of the apoptotic machinery in mammals. Our goal is to identify and characterize the mechanism regulating activation of the core apoptotic machinery downstream of DNA damage, which is the key to developing novel therapeutics for cancer. We hypothesize that the cellular metabolic state regulates the sensitization of cells to undergo apoptosis. This hypothesis is based on 1) our identification of several metabolic genes in a genome-wide RNAi screen in fly cells for direct regulators of caspases, 2) our observation that these genes modulate cellular NADPH levels, and 3) recent work from other labs demonstrating that NADPH levels regulate caspase activation and cell death. This proposal will focus on the mechanism by which these metabolic genes regulate the activation of caspases. The specific aims are to: 1. Test whether the metabolic genes directly regulate caspase activity by modulating NADPH levels in Drosophila Kc cells. Preliminary data show that reduced expression of the metabolic genes results in the increase in NADPH levels. We will determine whether (i) all genes identified in the RNAi screen modulate NADPH levels by an enzymatic assay for quantifying NADPH levels in cell extracts, (ii) up-regulation of the pentose phosphate pathway by over-expressing glucose-6-phosphate dehydrogenase inhibits caspase activity in response to DNA damage, and (iii) down-regulation of the pentose phosphate pathway by reducing expression of glucose-6-phosphate dehydrogenase promotes caspase activity in response to DNA damage. 2. Determine whether the metabolic genes regulate the post-translational modification of caspases by modulating NADPH levels in Drosophila Kc cells. We will (i) verify phosphorylation of Drone under normal conditions by radio-labeling recombinant Drone and test whether reduced expression of the metabolic genes alters this modification and (ii) verify S-nitrosylation of Drone under normal conditions by a modified Biotin- switch method and test whether reduced expression of the metabolic genes alters this modification. 3. Examine whether the metabolic genes regulate Diapl-mediated inhibition of Drone. We will test whether reduced expression of the metabolic genes alters the interaction between Diapl and Drone by immunoprecipitation assays. This proposal has broad implications in understanding cancer biology, which is characterized by altered apoptosis and glucose metabolism, and biology of aging, which is characterized by increased oxidative stress and apoptosis.